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or other changes. A massage of the tail was performed during palpation to stimulate local blood circulation during the first three postoperative days.
3.1.3.5 Sample harvesting and evaluations for “Direct OSSI” experimental model
The animals were sacrificed under general anesthesia with sodium pentobarbital (Nembutal, 40 mg/kg body weight, i.p.). We sacrificed 21 animals after 4 weeks, 21 animals after 8 weeks, 7 animals after 12 weeks, 14 animals after 16 weeks. Since we did not have preceding data with the presently developed methodology, for sample size calculation we used the pull-out evaluation data at the 4th and 8th weeks’ endpoints. At these endpoints we had 14 animals per group. Then we used the G*Power-free software (University of Dusseldorf, http://www.gpower.hhu.de/en.html). The α-error probe was 0.05, the power was 0.8, the allocation ratio N2/N1 was 1 and the effect size was counted as 2.87. Based on this calculation, we applied sample size n=7 in consecutive experiments.
The samples were used for either biomechanical (RFA and pull-out test) or for structural (micro-CT and histomorphometry) analysis. The tail was ligated at the bottom to control bleeding, then C3-C4 vertebrae were separated from the tails through surgical cutting the joint between C3-C2 vertebrae. The C3 vertebrae were used as healthy controls for C4 in histomorphometric and micro-CT analyses. For biomechanical evaluation, soft tissues were removed and the vertebrae were kept in 0.9% NaCl solution at 4°C until evaluations (from 12 to 48 hours). For structural analysis, the samples were fixed in a 10% buffered formaldehyde solution.
Based on the in vitro results, we set a complex evaluation protocol to analyze the interosseous implant anchorage in the bone tissue using combined biomechanical and structural methods. The biomechanical evaluation of osseointegration was performed applying RFA and pull-out tests, both on the same samples (Figure 10). The structural analysis was carried out by micro-CT and histomorphometry using the same samples.
43 3.1.3.5.1 Biomechanical evaluations
The two biomechanical tests were completed on the day of harvesting. We first performed RFA and then the pull-out test. 14-14 animals were tested at 4 and 8 weeks, while 7-7 animals were evaluated at 12 and 16 weeks.
3.1.3.5.1.1 RFA
A non-invasive (RFA) analysis was performed according to the previously described approach in the in vitro part. The RFA was recorded in 4 perpendicular directions, 5 times per direction (Figure 10.A). Then the average value of these 20 ISQ values was used to describe the stability of the particular implant.
3.1.3.5.1.2 Pull-out test
After the non-invasive evaluation by RFA, the axial extraction force was used to evaluate secondary implant stability. For the pull-out testing, we used a tensional test machine Instron 5965 (Instron®, USA) in collaboration with the Department of Materials Science and Engineering, Budapest University of Technology and Economics, Budapest, Hungary (personally with Dávid Pammer).
Measurements were done according to the following steps: a) the hook was screwed into the head of the implant. Then a thin stainless-steel cable (Ø1.5 mm) was pulled through the hook-head to provide an appropriate grip for the measuring device; b) after that the PUF block was fixed with a metal bracket to the plate of the Instron 5965 and the instrument was balanced, the implant was steadily pulled along the vertical axis until extraction (Figure 10.B). The maximal pull-out force (N) represents the strength of primary or secondary stability in the vertical axis. The pull-out test was applied in accordance with the ASTM F543 - 17 (American Society for Testing and Materials, Standard Specification and Test Methods for Metallic Medical Bone Screws) (ASTM, 2017). Its Annex A3 contains directives for the determination of pull-out test measurement parameters. Only one peak extraction value was detected for each implant.
44 3.1.3.5.2 Structural analyses
Twenty-one specimens (n=7 animals per group) were used for structural analysis such as micro-CT and histomorphometric analysis. The evaluation endpoints for structural analysis were at weeks 4, 8 and 16.
3.1.3.5.2.1 Micro-CT analysis
Before histological testing, we performed a 3D radiographic data acquisition to detect the structural basis of implant stability in the reconstructed 3D images (1172 SkyScan micro-CT, Bruker, USA). The device has an X-ray source from a sealed micro focus X-ray tube with a spot size of 8μm. In the present work an Al+Cu filter (Al 1.0 mm and Cu 0.05 mm) was used. The implant samples with bone were scanned at 360° rotation at 0.3 degree rotation step at 80kV, 124mA, 4598ms exposure time with an isometric voxel size of
Figure 10.
Biomechanical evaluations of osseointegration of longitudinally placed implant into rat tail caudal vertebrae.
A. Evaluation of implant stability with resonance frequency analysis (RFA) using SmartPeg 62 transductor, which was screwed into the implant head directly. B. Axial extraction force measurement algorithm, with sample positioning.
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12μm. For the reconstruction of raw images, a cone-beam volumetric algorithm was used with the NRecon V1.6.10.1 software (Bruker, USA). Measurements were performed within a certain region of interest (ROI) in the reconstructed images using the software CTAn V1.14.4.1+ (Bruker, USA) (Cha et al., 2009). The described protocol for scanning and reconstruction was specially designed and optimized to our experimental conditions in order to overcome the x-ray scattering on the metal surface.
The scanned samples were evaluated in 2D and 3D perspectives with task lists developed for this purpose in the CTAn software (Figure 11.A). The 2D analysis was done on slices from the 3D reconstructed sample. The calculated intersection surface/tissue surface ratio (i.S/TS) was used in the 2D analysis for characterizing the bone to implant contact. Based on the manufacturer’s instruction (1172 SkyScan micro-CT, Bruker, USA) and our calibration process, we chose the 12 pixel-wide dilation length around the implant for determining the intersection surface value expressed in percentage.
For bone volume assessment, a 38-voxel (0.461 µm) thick cylindrical volume of interest was selected around the titanium implant (Cha et al., 2009; Chang et al., 2013; Song et al., 2013). The manual global threshold method was used for the segmentation of new bone visualization. For determining the percentage of bone volume value, bone volume/tissue volume ratio was calculated (BV/TS). These studies were performed in collaboration with the Department of Oral Diagnostics of Semmelweis University.
3.1.3.5.2.2 Histology and histomorphometry
After micro-CT measurements, the samples were chemically fixed and embedded as previously reported (Liu et al., 2007). The sample processing was as follows: fixation in 10% buffered formalin; than the specimens were dehydrated in an ascending series of alcohol concentrations (50%–99%) and finally embedded in autopolymerizing methyl methacrylate resin (Wako Pure Chemical Industries Ltd., Osaka, Japan); afterwards the undecalcified tail vertebra specimens were cut using a diamond saw (SP 1600; Leica Microsystems, Wetzlar, Germany); then the received sections were adhered to the Teflon slides, and successively ground to a thickness of ~80 μm. The slices were then surface-stained with McNeal’s Tetrachrome, basic Fuchsine and Toluidine Blue O (Schenk RK,
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1984) for the histomorphometric analysis. The bone-implant contacts (BICs) were then analyzed under a light microscope with 4x and 10x magnification.
The bone to implant contact (BIC) evaluation was done using all the images. The BIC values of each sample were measured, and the average of the group was received.
The BIC measurements were performed manually. On each histological slide the same ROI was chosen (Figure 11.B). The ROI for BIC assessments was considered to be the intrabony 1.3 mm wide implant body part with parallel walls. The perimeter of the total ROI was measured for each sample, which was the total area for possible BICs. Also, for each sample, the length of individually-formed direct bone contacts to the implant surface was measured. According to the two measurements, BIC ratio was calculated for each sample individually. For that purpose, the total BIC length was divided by a sum of the established BICs. Based on that, we received the BIC ratios. Then the percentage was calculated from the ratio. Consequently, the average data were calculated for each healing period.
Figure 11.
Structural evaluations of longitudinally placed implant into rat tail caudal vertebrae.
A. Micro-CT capture of the